ISSN (Print) 0023-4001 ISSN (Online) 1738-0006

Korean J Parasitol Vol. 57, No. 4: 439-444, August 2019 ▣ BRIEF COMMUNICATION https://doi.org/10.3347/kjp.2019.57.4.439

Kudoa ogawai (: Kudoidae) Infection in Cultured Paralichthys olivaceus

1,2 1,2 1,2 1,2, Sang Phil Shin , Chang Nam Jin , Han Chang Sohn , Jehee Lee * 1Department of Science, Jeju National University, Jeju Self-Governing Province 63243, Korea; 2Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Korea

Abstract: Since Kudoa septempuntata was identified as a causative agent of food poisoning associated with raw olive flounder Paralichthys olivaceus, interest and concern regarding the parasite have increased. However, there have been no investigations or reports of other Kudoa species infecting the fish (except for K. paralichthys, which infects the brain) in Korea. We found cysts filled with myxospores of Kudoa species in muscles of cultured olive flounder specimens and identified these to the species level. Mature spores were quadrate, measuring 8.7± 0.5 µm in length, 9.2± 0.4 µm in thick- ness, and 12.9± 0.6 µm in width. The spores containing 4 polar capsules had a length of 2.1± 0.2 µm and a width of 1.8± 0.3 µm. The partial 18S and 28S rDNA of isolates showed 99-100% similarities with K. ogawai. Using these morpho- logical and molecular analyses, the species was identified as K. ogawai. This study is the first report of K. ogawai infection in cultured olive flounder in Korea.

Key words: Kudoa ogawai, olive flounder, identification, foodborne disease

Myxozoans are parasites that complete a life cycle through flounder cultured in Jeju island and has isolated another Ku- vertebrate (mainly fish) and invertebrate (mainly ) doa sp. from olive flounder muscle. The aim of the present hosts [1]. The genus Kudoa contains myxosporeans with 4 or study was to identify the Kudoa sp. using morphological and more shall valves and polar capsules, and the parasites infect molecular analyses and to reveal differences between the pres- various fish species [2,3]. Some Kudoa spp. reduce the com- ent isolate and other Kudoa spp. known to infect olive floun- mercial value of fish by causing post-mortem liquefaction der. In addition, the information obtained from this study will (such as K. thyrsites, K. lateolabracis, and K. neothunni) [3-7], be applied to the inspection and identification of Kudoa spp. cyst formation in muscles (K. iwatai and K. amamiensis) [8,9], Olive flounder samples (n=2, 56.4±4.3 cm) were obtained and spinal deformation (K. yasunagai) [2,10,11]. In addition, from an olive flounder farm situated on Jeju island. The fish previous studies have reported the relationship between Kudoa samples were examined microscopically to detect K. septem- spp. and food-poisoning [12-14]. These findings have led to punctata spores in squash preparations, and cysts filled with increased interest and concern regarding Kudoa infection in another Kudoa sp. were found. The cysts were collected using commercially important fish. Olive flounder, Paralichthys oliva- forceps and were squashed on a glass slide. The spores were ceus, is an important fish species cultured in Korea, and there wet mounted and observed under a light microscope and pho- have been several reports of myxosporean infections (Entero- tographed at 400×or 1,000×magnification. Measurements of myxum leei, Parvicapsula anisocaudata, P. curvatura, and Sinuolin- myxospores were obtain from 20 spores using the ImageJ im- ea capsularis), including Kudoa spp. (K. septempunctata and K. age processing software (available at http://rsb.info.nih.gov/ paralichthys), in the fish [15-19]. The Fish Vaccine Research ij/) according to Lom and Arthur’s criteria (1989) [20]. Center has been monitoring the parasitic infections of olive The myxospores were collected and passed through a 40 µm cell strainer. DNA was extracted from partially purified para-

Received 8 May 2019, revised 30 July 2019, accepted 31 July 2019. sites using the AccuPrep Genomic DNA Extraction Kit (Bi- • Corresponding author ([email protected]) * oneer, Daejeon, Korea) following the manufacturer’s instruc- © 2019, Korean Society for Parasitology and Tropical Medicine tions. Portions of 18S and 28S rDNA were amplified by PCR This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) using a combination of primers that we (28S-KO2000F: 5′ which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. -CTGTCCGTACCGAATCCG-3′) and other groups (18S-18e: 5′

439 440 Korean J Parasitol Vol. 57, No. 4: 439-444, August 2019

-CTGGTTGATCCTGCCAGT-3′, 18S-MyxospecF: 5′-TTCT- myxospores were quadrate in the apical view and had 4 equal GCCCTATCAACTWGTTG-3′, 18S-MyxospecR: 5′-GGTTTCN spore valves, each containing one polar capsule with a round- CDGRGGGMCCAAC-3′, 18S-ERIB10: 5′-CTTCCGCAG- ed peripheral edge. In addition, the appearance of the 4 polar GTTCACCTA-3′, 28S-Kt28SF: 5′-CAAGACTACCTGCTGAAC-3′, capsules was similar to a 4-leaf clover (Fig. 1D). In side view, 28S-NLF1050: 5′-AATCGAACCATCTAGTAGCTGG-3′, 28S- spores were rounded triangle-shaped, and prominent apical NLR1270: 5′-TTCATCCCGCATCGCCAGTTC-3′, 28S- projections were observed (Fig. 1E). The spores measured NLR1694(mo): 5′-GTTAGGCAATGGCTTAGGACC-3′, 28S-3R: 8.7±0.5 µm (8.3-9.6 µm) in length, 9.2±0.4 µm (8.4-10.0 5′-GAGCACTGGGCAGAAATC-3′, 28S-NLR3113(mo): 5′-GTC- µm) in thickness, and 12.9±0.6 µm (11.5-13.9 µm) in width. TAAACCCAGCTCACGTTC-3′, and 28S-NLR3284: 5′-TTCT- The polar capsule length was 2.1 ±0.2 µm (1.7-2.5 μm) and GACTTAGAGGCGTTCAG-3′) have designed [2,16,21-25]. PCR polar capsule width was 1.8±0.3 µm (1.4-2.5 µm) (Table 1). products were treated with the AccuPrep Genomic PCR Purifi- The species isolated in the present study was then compared cation Kit (Bioneer) to remove excess primers and dNTPs, and with 8 Kudoa spp. that have been previously reported in olive were directly sequenced using BigDyeTM Terminator v3.1 in flounder. Kudoa ogawai was the most similar to the isolated an ABI 3730xl Sequencer. The 18S and 28S rDNA sequences species in terms of measurements and shape of spores, num- of isolates were compared in the GenBank database using the ber of valves per spore, apical projection, and infection site. Basic Local Alignment Search Tool (BLAST) search engine to Kudoa paralichthys and K. shiomitsui showed similarities to the find sequences with a high degree of similarity. Multiple align- isolate in terms of spore shape and number of valves per ments of 18S rDNA sequences were made using Clustal X 2.0 spore. However, they are smaller than the isolate and have a [26] with 7 homologous sequences from Kudoa spp. and one different infection site (brain and vs muscle). In addi- sequence from E. leei (outgroup) reported in olive flounder tion, they have small projections that are not easily observed, (except K. lateolabracis). The phylogenetic tree was constructed while the isolate has distinct apical projections. Although K. using the neighbor-joining method in MEGA 7 [27]. lateolabracis and K. thyrsites had similarities number of valves Oval cysts 1 mm in length approximately, were observed in per spore and share the same infection site as the isolate, they the squashed muscle sample (Fig. 1A). Cysts were shorter in have distinct spore shapes with one large polar capsule. Kudoa length and thicker than the pseudocysts of K. septempunctata septempunctata and K. igami have similar spore measurements that are usually detected, and the myxospores were also differ- and the same infection site as the isolate. However, they have a ent in form from those of K. septempunctata (Fig. 1B, C). The different spore shape (stellate vs. quadrate) as well as a differ-

A B C

Fig. 1. Kudoa ogawai found in the muscle of Paralichthys olivaceus. (A) Cyst and (B) myxospores of K. ogawai. Scale bar= 100 and 20 µm, respectively. (C) Myxospores of K. septempunctata. Scale bar= 20 µm. (D) Fresh spores of K. ogawai in apical view and (E) side view. Arrows and arrowheads show the polar capsule and the apical pro- jections of spores, respectively. Scale D E bar= 10 µm. Shin et al.: Infection of Kudoa ogawai 441

ent number of valves per spore (6 or 7 vs 4). In addition, their apical projections are not prominent as those of the isolate [4] [4] [28] [29] [28] [19] [11] [30] study Present Present (Table 1) [4,11,19,28-30]. References Partial sequences of the 18S rDNA (1,650 bp) and 28S

site rDNA (3,395 bp) were obtained from the isolate and had 100 Brain Brain Heart Muscle Muscle Muscle Muscle Muscle Muscle Infection and 99.9% similarities with the 18S (KX163082; 1,631/1,631) and 28S rDNA (KX163083; 3,318/3,323) sequences of K. oga- wai isolated from olive flounder in Japan, respectively. Unfor- - - - - + +

Apical tunately, there is no 18S rDNA sequence of K. paralichthys projections Tiny projections Small projections Small projections available on GenBank. In addition, the 18S rDNA sequence of K. lateolabracis reported from olive flounder could not be found, thus the sequence of K. lateolabracis isolated from Chi- nese sea bass was used to construct the phylogenetic tree. The

phylogenetic tree was divided into 4 groups and the isolate Spore shape Spore was clustered with K. ogawai (Fig. 2). Based on the morpho- polar capsule (4) polar capsule (4) (Np. of valves per spore) Quadrate (4) Quadrate (4) Stellate (6/7) Quadrate with unequal Quadrate with unequal Stellate (6) Quadrate (4) Stellate (7) Quadrate (4) logical and molecular comparisons, the myxospores were identified as K. ogawai and the sequences were deposited in GenBank (accession numbers MK850343 and MK850346, re- - - width 2.2-2.8 1.0-1.4 2.2-2.9 1.3-1.7 spectively). (1.4-2.5) 1.8± 0.3 Paralichthys olivaceus Polar capsule 1.5-2.5 Large) 1.5-2.0 (Small) 2.5-3.5 (Large) 1.5-3.0 (Large) Korea and Japan have a food culture that involves eating raw fish, which increase the risk of food poisoning. Kudoa septem- punctata has been reported as a causative agent of food poison-

- - ing after ingesting raw olive flounder [12,31]. Kawai et al. [12] length 3.7-5.3 1.9-2.7 3.4-4.3 1.9-3.9 (1.7-2.5) 2.1± 0.2 reported that oral administration of K. septempunctata spores Polar capsule 3.0-4.0 (Small) 4.0-5.9 (Large) 4.0-5.9 (Large) 2.0-4.0 (Large) elicited accumulation of fluid in the intestine and produced watery diarrhea in suckling mice. They also showed that the direct feeding of olive flounder meat containing K. septempunc- spp. reported in olive flounders spp. reported tata spores as well as purified spores cause vomitting in house width Spore Spore 8.4-9.9 (Max) 5.9-6.9 (Min) 7.9-11.9 (Max) 6.9-7.9 (Min) 7.4-8.9 9.4-13.9 8.8-10.3 Kudoa 12.9± 0.6 12.3± 0.7 10.8± 0.6 11.1-13.1 (11.5-13.9) musk shrews. Ohnish et al. [31] revealed that K. septempunctata (Kasai et al., 2016 [30]). (Hsieh et al., 1984 [11]). sporoplasm plays an important role in mediating the toxicity of K. septempunctata. In addition, previous studies have investi- sp. at the original description (Yokoyama et al., 2004 [4]). sp. at the original description (Yokoyama gated the epidemiological relationship between K. septempunc- tata ingestion and food poisoning in Korea and Japan [13,32]. Spore Spore 8.9-10.0 9.9-12.9 6.3-7.7 7.1-10.2 7.4-8.7 9.2± 0.4 9.9± 0.7 9.6± 0.6 Lateolabrax thickness (8.4-10.0)

12.9-17.8 Iwashita et al. [32] reported 3 clinical cases related to K. sep- Lateolabrax japonicus Stephanolepis cirrhifer tempunctata ingestion in the Mie prefecture of Japan, and Kim et al. [13] also reported on the epidemiologic characteristics of

isolated from isolated from 16 outbreaks of foodborne illnesses caused by K. septempuncta- sp. (present isolate) with the other sp. (present isolated from isolated from isolated from isolated from Spore Spore length ta in the Gyeonggi Province of Korea. However, some studies 7.9-8.9 5.4-6.9 6.9-8.9 4.5-6.0 4.3-7.3 6.2-7.4 (8.3-9.6) 8.7± 0.5 9.5± 0.5 7.0± 0.3

Kudoa have suggested that there is no relationship between food poi- soning and ingesting olive flounder infected by K. septempunc- K. yasunagai K. shiomitsui K. lateolabracis tata [33-35]. Jang et al. [33] showed that suckling mice inocu-

a 6-7 b c lated with 5×10 K. septempunctata spores did not show

Comparison of symptoms of diarrhea, and no pathological change in intes- sp. tine of the mice. Ahn et al. [34] came to similar results in study Data provided from from Data provided Data provided from from Data provided from Data provided Species Kudoa K. ogawai K. septempunctata K. lateolabracis K. thyrsites K. igami K. paralichthys K. yasunagai K. shiomitsui Table 1. Table b c a using adult mice. In addition, they exhibited that K. septem- 442 Korean J Parasitol Vol. 57, No. 4: 439-444, August 2019

Fig. 2. Phylogenetic tree generated by MEGA 7, based on the aligned partial 18S rDNA sequences of Kudoa sp. newly obtained from the present study and Kudoa spp. previously reported in olive flounder. leei was set as the outgroup and the bootstrap values (1,000 replicates) are shown at the nodes on the neighbor-joining tree. The present species is indicated by an arrow and is clus- tering with K. ogawai. punctata spores did not cause inflammation on Caco-2 cells sites. Thus, more studies investigating other myxosporeans (es- [35]. It is therefore up for discussion whether K. septempunctata pecially parasites that infect muscle) and developing control causes food poisoning or not, however a few cultured olive methods against parasite infections should be conducted. flounder are undeniably infected by the parasite. In the present study, Kudoa sp. was discovered to infect the ACKNOWLEDGMENTS muscle of cultured olive flounder, and the isolate was identi- fied as K. ogawai based on morphological and molecular com- This work was supported by the Basic Science Research Pro- parisons. There have been no studies revealing the relationship gram through the National Research Foundation of Korea between K. ogawai and food poisoning, however, there have (NRF) funded by the Ministry of Education (2016R1D- been several reports that other myxosporeans (not K. septem- 1A1B03932598) and is part of the project titled ‘Fish Vaccine punctata) may cause foodborne diseases [14,36,37]. Kawase et Research Center’, funded by the Ministry of Oceans and Fish- al. [36] reported that K. hexapunctata genes were detected in eries, Korea. the feces of a patient exhibiting diarrhea and vomiting after eating raw juvenile Pacific bluefin tuna, Thunnus orientalis, and CONFLICT OF INTEREST Ohnishi et al. [14] also detected the genes and spores of K. iwatai from Rock bream (barred knifejaw), Oplegnathus fascia- The authors declare no conflict of interest related to this tus, and Blackhead seabream, Acanthopagrus schlegelii, associat- study. ed with gastrointestinal disease. In addition, Unicapsula seriolae is also suspected to be a parasite associated with an unidenti- REFERENCES fied foodborne disease of caused by ingestion of Greater am- berjack, Seriola dumerili [37]. Screening for K. septempumnctata 1. Chang ES, Neuhof M, Rubinstein ND, Diamant A, Philippe H, is carried out on cultured olive flounder by the Korean govern- Huchon D, Cartwright P. Genomic insights into the evolution- ment, however other myxosporeans containing other Kudoa ary origin of within . Proc Natl Acad Sci USA 2015; 112: 14912-14917. spp. that infect the fish are not investigated. Because Kudoa 2. Whipps CM, Grossel G, Adlard RD, Yokoyama H, Bryant MS, spp. have a low host specificity [38], there is a possibility that Munday BL, Kent ML. Phylogeny of the multivalvulidae (Myxo- cultured olive flounders are infected by other Kudoa spp. (or zoa: Myxosporea) based on comparative ribosomal DNA se- myxosporeans) related with foodborne illness. In addition, quence analysis. J Parasitol 2004; 90: 618-622. most consumers do not want to eat fish infected by any para- 3. Eiras JC, Saraiva A, Cruz C. Synopsis of the species of Kudoa Shin et al.: Infection of Kudoa ogawai 443

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